Nontypable Haemophilus influenzae is a common cause of localized respiratory tract disease, including otitis media, sinusitis, bronchitis, and pneumonia. In addition, this organism causes serious systemic disease, such as meningitis, endocarditis, and septicemia. The initial step in the pathogenesis of nontypable H. influenzae disease involves colonization of the upper respiratory mucosa. We have identified an H. influenzae serine protease called Hap, which facilitates intimate interaction with epithelial cells and extracellular matrix proteins and also promotes bacterial aggregation and microcolony formation. Based on our in vitro results, we speculate that Hap plays an important role in the process of colonization. Hap belongs to the growing family of autotransporter proteins and is synthesized as a precursor protein with 3 functional domains, including an N-terminal signal sequence, an internal protease domain with adhesive activity (Haps), and a C-terminal outer membrane domain with translocator activity (HapBeta). Ultimately, Hap undergoes autoproteolytic cleavage, with extracellular release of Haps. In recent work, we demonstrated that Hap mediated adherence and microcolony formation are potentiated by a host protein called secretory leukocyteprotease inhibitor (SLPI). This protein is present in respiratory secretions and inhibits Hap autoproteolysis, resulting in accumulation of surface-associated Haps. In the present proposal, we will focus on Hap-mediated adherence and microcolony formation. In Aim 1, we will solve the crystal structure of Haps and define the interactive surfaces involved in adherence and microcolony formation. In Aim 2, we will examine the ability of microcolonies to resist killing by cationic peptides, to evade macrophage phagocytosis, and to enhance persistence in the chinchilla otitis media model. In Aim 3, we will characterize the relationship between respiratory viral infection and Hap-mediated adherence and microcolony formation, concentrating on the role of SLPI. From a practical perspective, the proposed studies may facilitate efforts to develop novel strategies forthe treatment and prevention of H. influenzae disease. Perhaps more importantly, they may provide general insights into host-microbe relationships and expand our understanding of microbial biofilms.